Screening Of Ester-producing Yeast Of Cane Whisky And Study On Its Fermentative Process

Whisky is one of four types of most famous distillated spirit and plays an important role in the international trade of wine. At present, the report about Whisky is few in our country and the study of Whisky with cane juice as main material haven't been reported. So, to expand civil research, this paper aimed to several technical key points of cane Whisky and mainly carried on a series of study work in screening and breeding of strains.1. ADY was mutated with ultraviolet radiation (UV) and those strains that grew on the plate containing TTC with white colonies were screened out as positive mutants. The selected mutant, YS-5-36, was suitable for fermenting cane juice. The optimal fermentative conditions was obtained by using single-factor experiments as follows: the initial pH was 5.0, the temperature was 28℃, inorganic nitrogen source was (NH₄)₂SO₄ or NH₄Cl.2. The strains from distiller's yeasts of Luzhou-flavor liquors were cultivated enrichly and the single colonies were separated by selected plates. Then, all these strains with the typical yeast colonies were identified into three types including Hansenula, Brettanomyces and Dekkera. Y2-7 that had the highest total esters was selected and its yield of total esters was 0.221g/L. Through the cross experiment, the optimal condition of producing esters was obtained as follows: the volume ratio of 25% malt extract and cane juice was 1:4, the temperature was 24℃, the time was 7 days. And the yield of total esters reached 2.142g/L.3. Y2-7 was mutated with three methods. The optimal conditions of three methods were obtained and the ability of producing alcohol and esters was compared. The optimal condition of UV was as follows: the power of ultraviolet light was 15W, the distance was 30 cm, the time was 40s. The results showed that the yield of total esters reached 2.219g/L and increased by 3.7% than parent strain after fermentation. The optimal condition of DES was that the quantity of 5% DES was 1.2 ml and the time was 50 minutes. The results showed that the yield of total esters reached 2.228g/L and increased by 4.2% than parent strain. The optimal condition of UV+DES was that the time was 40s with UV at first and then the treatment time was 30min with 1.2 ml 5% DES. And the yield of total esters reached 2.247g/L and increased by 5.1% than original strain.4. YS-5-36 and Y2-7 were fused by inactivated protoplast fusion. The optimal condition of protoplast formation and regeneration were obtained. The yeasts were cultured 8 hours at first, then pre-treated 30 minutes at 30℃with 0.05mol/L EDTA and 0.5mol/Lβ-sulfydryl ethanol and treated 70 minutes with 1.5% lysozyme at 30℃. The fusants were obtained by PEG-induced fusion between UV-inactivated protoplasts of YS-5-36 and heat-inactivated protoplasts of Y2-7. And the fusion rate was 1.85×10^-6. The three fusants, SH-6, SH-9 and SH-13, were selected by selected medium. The yield of total esters of SH-13 reached 2.388g/L, which increased by 6.4% than parent strain.5. The optimal condition of producing esters of SH-13 was decided by the cross experiment. It was that the volume ratio of 20% malt extract and cane juice was 1:3, the temperature was 24℃, the time was 7 days and the initial amount of strains was 6%. And the yield of total esters of SH-13 reached 2.427g/L.6. The distilled spirits matured by adding the burnt oak pieces. The best immersing condition, which was obtained by gas chromatography to analyze main esters was that the second distilled spirits was immersed three months at 20℃by adding 5% oak pieces.Under this condition, Whisky complied with the national standard. It had a high degree of clarity without suspension, deposits and unpleasant smells. Its color was golden and it had a rich aroma of malt and cane as well as a harmonious flavor of oak, which made it have strong acceptability and adaptability.